The invention is related to a novel potassium channel protein KCNQ5 which is considered as a target for diseases of central nervous system and cardiovascular system. The protein is used as a screening tool for identification of compounds which improve symptoms of diseases of the central nervous system and cardiovascular system.
Voltage-dependent potassium channels are key regulators of the resting membrane potential and modulate the excitability of electrically active cells like neurons and cardiac myocytes. Several classes of voltage-dependent K+ channels have been cloned and probably all form oligomeric proteins through the assembly of four xcex1-protein subunits. The tetrameric pore complex further can interact with auxiliary subunits that enhance and/or modify currents mediated by the pore-forming xcex1-subunits.
The KCNQ family of voltage-dependent K+ channels originally was established by positional cloning of the KCNQ1 gene (KvLQT1) which encodes a K+ channel protein with six transmembrane domains and a characteristic pore region. So far the KCNQ family consists of four members all of which are associated with human diseases. KCNQ1 functionally interacts with KCNE1, a small xcex2-subunit protein with a single transmembrane domain, to generate the slowly-activating delayed-rectifier IKs current of cardiomyocytes. Inactivating mutations in both subunits result in the prolongation of the cardiac action potential and an increased risk of ventricular arrhythmias in patients with long QT-syndrome (LQTS). KCNQ1 and KCNE1 also are found in the inner ear and some loss of function mutations of KCNE1 and KCNQ1 are associated with hearing loss. In intestine, KCNQ1 probably associates with the structurally related KCNE3 protein to generate different K+ channels. The KCNQ1/KCNE3 channel complex possibly represents the basolateral cAMP1-regulated K+ conductance in colonic crypts probably important for apical cAMP-stimulated chloride secretion and that is involved in secretory diarrhea and cystic fibrosis.
KCNQ2 and KCNQ3 are expressed in the brain and colocalize in various brain areas (Biervert, C. et al.; Science 279, 403-406, 1998). Whereas KCNQ2 generates K+ currents very similar to KCNQ1 (Schrxc3x6der, B. C. et al.; Nature 396, 687-690, 1998), KCNQ3 alone produces only small currents (Wang, W.-P. et al., J. Biol. Chem. 273, 19419-19423, 1998). Coexpression of both KCNQ2 and KCNQ3 resulted in currents that were at least 10-fold larger than that of KCNQ2 alone (Wang, H.-S. et al.; Science 282, 1890-1893, 1998), suggesting that KCNQ3 facilitates expression of KCNQ2 subunits, by formation of a heteromeric complex of KCNQ2 and KCNQ3 subunits. The genes encoding KCNQ2 and KCNQ3 have been cloned due to their linkage to a form of epilepsy in human infants and loss of function mutations have been identified in both genes in patients with benign familial neonatal convulsions (BNFC) (Sigh, N. A. et al.; N. Genet. 18, 25-29, 1998; Charlier, C. et al.; N. Genet. 18, 53-55, 1998)). Since epilepsy is due to an electrical hyperexcitability in the brain, KCNQs may have an important stabilizing role in the nervous system. Biophysical and pharmacological properties of KCNQ2/KCNQ3 currents are very similar to that of the native neuronal M-type K+ current that is also characterized by muscarinic modulation and that is thought to be a prominent regulator of neuronal excitability as well. Similar to the native M-current, KCNQ2 KCNQ3 channel activity is strongly reduced by muscarinic acetylcholine agonists and therefore it is now assumed that KCNQ2 and KCNQ3 subunits contribute to the native M-channel.
KCNQ4, another member of this gene family, is expressed in sensory outer hair cells of the cochlea and is mutated in dominant deafness. Interestingly, coexpression of KCNQ3 with KCNQ4 also increased current amplitudes, although to a far less extent than observed with KCNQ2/KCNQ3 coexpression. This raises the possibility that different KCNQ channels can combine to produce variants of M-currents in different parts of the nervous system.
The object of the invention was to identify a gene coding for another member of the KCNQ family and the protein for use as a marker for treatment of various diseases. Compounds modifying the activity of KCNQ5 potassium channels may be useful in the treatment of some forms of epilepsy and other neurological or cardiovascular disorders.
In a first embodiment, an isolated nucleic acid molecule encoding a KCNQ5 polypeptide comprising SEQ ID NO:2 is provided. The invention further relates to an isolated nucleic acid molecule that is a nucleic acid sequence comprising SEQ ID NO:1. In another embodiment, the invention relates to a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1, a nucleic acid sequence encoding SEQ ID NO:2; or a nucleic acid sequence that hybridizes under stringent conditions to one of the above; or a nucleic acid sequence that is complementary thereto.
In another embodiment, the invention provides an expression vector comprising an isolated nucleic acid molecule described above.
A further embodiment of the invention is a host cell, comprising the expression vector described above and a method of producing such a host cell.
The present invention also relates to an isolated polypeptide, with KCNQ5 activity, encoded by one of the above described nucleic acid molecules. Said protein may be produced by propagating a host cell harboring a recombinant vector including a DNA sequence encoding for an amino acid sequence or a polynucleotide sequence for
KCNQ5 in a growth medium suitable either for bacteria or eucaryotic cells depending on the host cell type. These propagated cells are then harvested by common biochemical methods, such as centrifugation or filtration, and processed to obtain crude cell extracts. These cell extracts are purified by methods used for protein purification, such as size exchange chromatography, ion exchange chromatography, affinity chromatography and others, to retrieve the protein of interest (KCNQ5) separated from other compounds of the cell lysates.
In one embodiment, this isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof. The fragment generally contains at least about 10 consecutive amino acids and preferably about at least 30 to 50 consecutive amino acids.
In an additional embodiment, the isolated polypeptide of the invention comprises an amino acid sequence encoded by a polynucleotide that is SEQ ID NO:1. In another embodiment, the polypeptide of the invention is encoded by a nucleic acid sequence that is 95% identical to SEQ ID NO:1; or is a nucleic acid sequence that hybridizes under stringent conditions to one of the above, wherein the encoded polypeptide generates voltage-dependent, slowly activating K+-selective currents that are insensitive to the K+ channel blocker TEA and display a marked inward rectification at positive membrane voltages.
A further embodiment discloses a fusion protein comprising a polypeptide consisting of SEQ ID NO:2.
In an additional embodiment, this invention relates to an antibody that selectively binds KCNQ5. Further, the invention discloses a method of detecting a KCNQ5 polypeptide in a biological sample comprising contacting a detectably labeled antibody that selectively binds KCNQ5 with the biological sample and detecting the binding of the antibody with the KCNQ5 polypeptide in the biological sample.
In another embodiment, a cell culture for screening agonists and antagonists of KCNQ5 comprising cells that express KCNQ5 is disclosed. An example of such cells that express KCNQ5 are oocytes transformed with a nucleic acid molecule that transcribes SEQ ID NO:2.
A further embodiment of the invention is a method of screening for a compound that modifies the activity of KCNQ5. This modification may be, for example, inhibitory. In the method, a cell culture described above is provided, along with a compound to be screened. The compound may be a polypeptide, chemical compound, antibody, biological agent, antisense molecule, polynucleotide, oligopeptide, natural compound, or secondary metabolite. The activity of KCNQ5 without the compound is assessed by measuring K+-selective currents from a single channel, single cell, or a membrane patch; or by measuring a signaling event, such as an ion flux. The compound is then added to the cell culture and incubated. The activity of KCNQ5 is then reassessed and compared to the activity without the compound to determine whether said compound modifies said activity of KCNQ5.
In an additional embodiment, a method of detecting a nucleic acid sequence encoding SEQ ID NO:2 in a biological sample comprising contacting a labeled nucleic acid probe that hybridizes with the nucleic acid sequence with the biological sample under conditions wherein the probe hybridizes with the nucleic acid sequence and detecting the hybridization of the probe to the nucleic acid sequence in the sample is provided.
In an additional embodiment, a kit comprising one or more containers, wherein at least one container contains a detectably labeled antibody that selectively binds a KCNQ5 polypeptide is provided. A kit comprising one or more containers, wherein at least one container contains a detectably labeled nucleic acid probe that hybridizes with a polynucleotide encoding SEQ ID NO:2 is also provided.
In a further embodiment, transgenic nonhuman animals having a transgene encoding KCNQ5 are also described.